Field of the Invention
The present invention relates to an apparatus for structured illumination, and in particular for confocal observation of a specimen, wherein the specimen particularly comprises biological and/or chemical substances.
Description of Related Art
Known structured illumination microscopes comprise an illumination device which can be e.g. a laser. With the aid of the illumination device, an illumination beam is generated which first will be incident onto a mask which can comprise a plurality of openings, which may be shaped e.g. as slots or pinholes. Via an objective device, the mask will be imaged in the specimen. In this process, an image of the mask is generated in a clearly defined plane of the specimen. The illumination beam causes a reaction in the specimen, thus generating detection beams. Said reaction can be e.g. a fluorescence excitation of markers included in the specimen, or also a simple reflection of the illumination beams. Several uses of structured illumination are known in the art, including the observation of transport processes, which is achieved by exciting or bleaching markers in particular regions of the sample, and then observing the spatial distribution of light emitted from the sample over a period of time, whereby the spatial distribution varies according to a movement of the markers in the specimen which is caused by transport processes under study. Another use of structured illumination is confocal observation of the sample, as detailed below.
To achieve confocal observation of the specimen, the detection beams emitted by the specimen will be returned, again via said objective device, to the mask and will pass through the corresponding openings of the mask. With the aid of a beam splitter device, the detection beams, after passing through the mask device, will be guided out of the optical path toward a detection device such as e.g. a CCD sensor. It is known in the art that this confocal arrangement of mask openings in both, the illumination and the detection path, will effectively suppress light originating from specimen regions outside the objective device's focal plane, providing the benefit of creating an optical slice through the sample.
When using mask devices with very small openings, the problem exists that only small amounts of illumination radiation will pass through the mask device and reach the specimen. A known approach to cope with this problem is the provision of microlenses upstream of the openings of the mask device, which microlenses will bundle the beams and resp. guide them toward the openings of the mask device. In this arrangement, the beam splitter device for outcoupling the detection beams is disposed between the mask device and said beam-bundling device, e.g. the lenses.
This known arrangement has several disadvantages and limitations. Precisely collimated light is required to be incident onto the microlenses, in order to ensure that the focus of each individual microlens is positioned exactly at the location of the corresponding mask opening, both in the lateral and axial directions. In practice, this requirement limits the choice of light sources to lasers, which are costly. Further, particularly in movable mask devices such as e.g. Nipkow disks, it is difficult to obtain precise alignment between the individual microlenses and the mask openings, and to ensure that this alignment is maintained during movement—e.g. rotation—of the mask device. In practice this means that, in spite of the use of microlenses for the bundling of rays, a part of the illumination beams will not pass through the openings of the mask device. As a consequence, the transillumination intensity entering the specimen, and thus also the intensity of the detection beams issuing from the specimen, will be reduced. This leads to a reduction of the information obtained when examining the specimen. Furthermore, since the microlenses need to move in synchrony with the mask device, the size of the microlens array is typically required to be much larger than the field of view, resulting in increased cost of the microlens array.